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Previous studies have shown that a functional Ad 5 E1A protein from the 13 S mRNA is required during lytic infection for activation of early viral gene expression (Jones and Shenk, 1979b; Berk et al, 1979; Montell et al, 1982). The mechanism by which E1A exerts this regulatory function is still unclear and has been the subject of intense investigation (Persson et al, 1981a; Katze et al, 1981, 1983; Nevins, 1981; Shaw and Ziff, 1982). Most of these recent investigations have relied on the use of metabolic inhibitors such as cycloheximide to eliminate protein synthesis as a means of determining the role of viral and host proteins in the regulatory process. The results from these studies have been inconsistent. The purpose of this research project has been to re-examine the regulation of Ad 5 early gene expression without the use of drug inhibitors. In this study tsH1 cells, a mutant CHO cell line which at temperatures above 37°C are inhibited in protein synthesis, were used. At critical times during the course of wild type or host-range 1 infection of tsH1 cells, protein synthesis was inhibited and Early Region 4 expression was examined. In every case, efficient E4 expression was dependent on functional E1A protein and this requirement could not be replaced by simply inhibiting protein synthesis. The results are discussed in relation to models proposed to explain the regulation of Ad 5 Early Gene Expression. / Thesis / Master of Science (MS)

The phenomenon of residual inhibition, whereby the phantom sensation of tinnitus is suppressed following the presentation of a masking stimulus, has significant implications for understanding the neural basis of tinnitus itself. By using novel psychoacoustic techniques and three computer-based tools developed and applied specifically to measure tinnitus sensation and residual inhibition, a pattern emerges in which the depth and duration of tinnitus suppression relates to the center frequency of the band-passed noise masking stimulus. A correspondence between the region of hearing loss, the tinnitus spectrum and the masking stimuli most effective in suppressing tinnitus is revealed. These results suggest that cortical reorganization observed in animal models of tinnitus is not the principal basis of tinnitus, and provide a baseline for optimizing residual inhibition in individual cases and for further experiments. / Note: Pages 17-23 in this thesis were replaced with a citation due to copyright issues. / Thesis / Master of Science (MSc)

To evaluate continuous and sporadic nitrification inhibition at the HRSD Nansemond Wastewater Treatment Plant, which has a history of nitrification upsets, continuous sequencing batch reactors (SBRs) were operated to simulate the full-scale plant. Four reactors were operated in this study. One reactor was fed with raw influent (RWI) from the Nansemond Wastewater Treatment Plant (NP). Another was fed with NP primary clarifier influent (PCI), which includes the raw influent, as well as plant recycle streams and truck delivered septage, grease, and chemical toilet waste. The remaining two SBRs were fed with RWI from the VIP Wastewater Treatment Plant, which achieves reliable nitrification year-round. One of these VIP SBRs would remain a control at all times, while the other would be used to evaluate suspected inhibitors to nitrification. The first phase of this project was to determine whether NP was inhibited when compared to VIP, which would be ascertained through a comparison of nitrification performance. The next step was to determine whether the source of inhibition was an industry within the collection system or plant recycles and delivered wastes, which would be ascertained based on comparison of the NR RWI and NP PCI reactor performance. If nitrification performance was comparable between the two SBRs, then it would indicate that the source of inhibition is somewhere within the collection system, whereas if the NP PCI reactor was inhibited when compared to the NP RWI reactor, it would mean that the inhibition is a result of plant recycles or delivered wastes. The next phase would be to determine the specific source by either working back up the collection system or by testing the plant recycles and delivered wastes. After approximately 27 weeks of SBR sampling and monitoring, there was no statistical difference between nitrification rates in reactors A and B, and no signs of nitrification inhibition in either reactor when compared to the VIP control Simulation modeling of reactors A, B, and D (control) was performed with BioWin 3.1 (EnviroSim, Ltd.) as a means for comparison and to ensure reactors were performing as intended. Results suggest that there was some level of continuous inhibition for both NP RWI and PCI reactors, however no sporadic inhibition events were observed. It also appeared that the VIP RWI control reactor experienced some level of continuous nitrification inhibition, although BioWin modeling results indicated that both NP RWI and NP PCI were more inhibitory than VIP RWI. Conclusions drawn from modeling results conflict with those drawn from nitrification rate comparisons. Since solids retention time (SRT) was maintained at exactly 15 days for all reactors, it was assumed that a direct comparison of corrected maximum nitrification rates could be used to compare nitrification performance between SBRs, however the significantly higher influent COD, TKN, and TSS loading to the NP reactors resulted in higher nitrification rates when compared to the VIP RWI control reactors. This was confirmed with BioWin modeling, which also showed consistently higher nitrification rates for NP when compared to VIP RWI, however BioWin also showed that maximum specific growth rates for ammonia-oxidizing bacteria (?maxAOB) in NP RWI and PCI were consistently lower than the ?maxAOB for VIP RWI. This indicates that NP RWI and NP PCI are slightly inhibitory to nitrification, with ?maxAOB values between 0.65 and 0.75 days??, and the fact that both NP RWI and NP PCI are both inhibitory suggests that the source of inhibition is somewhere within the collection system. In a simultaneous study using the reactors fed with raw influent from the VIP Wastewater Treatment Plant, reactor C was spiked with aqueous Film Forming Foam (AFFF) such as that used in methanol feed facility fire suppression systems, while reactor D was left as a control. AFFF was initially added at a concentration of 20 ppm with no effect on either nitrification or denitrification performance. When increased to 40 ppm, the AFFF reactor experienced a complete loss of denitrification, while nitrification rates were not affected when compared with the control reactor. Reactor C took 31 days to fully acclimate to the AFFF feed and fully regain denitrification, and then exhibited no other performance problem throughout this acclimation period. This result was completely unexpected, appears to be repeatable, and is one of very few cases of selective denitrification (and COD uptake) inhibition, as opposed to more commonly observed nitrification inhibition. / Master of Science

Psychopathology is highly prevalent during childhood and adolescence and contributes to a variety of negative outcomes. Attempts to identify etiological factors which contribute to the development of psychopathology in youth have considered the Goodness of Fit between children's temperaments and the behaviors exhibited by their parents (Chess and Thomas, 1999; Zuckerman, 1999). Many studies have demonstrated that the interaction of children's behavioral inhibition and certain parenting behaviors influences children's psychological outcomes. However, the ability to draw firm conclusions from these studies is severely limited by methodological weaknesses. In the current study, data were analyzed from 253 youth (46% male) who completed assessments at 2-years (N=167), 3-years (N=144), 4-years (N=134), 6-years (N=110), and 9-years of age (N=192), and during adolescence (N=78; mean age=14.08 years). Measures of child behavioral inhibition, maternal warmth and control, and child psychopathology were gathered at each time point. Cross-sectional and longitudinal analyses were conducted to explore the moderating effect of maternal warmth and control on the relationship between child shyness and child/adolescent internalizing and externalizing symptoms. With a few exceptions, child shyness significantly predicted child internalizing symptoms at each time point, while maternal warmth and control, and their interaction with child shyness, did not predict child internalizing or externalizing outcomes. Longitudinally, the slope of shyness across childhood significantly predicted adolescent internalizing symptoms. The moderating effect of maternal parenting on this relationship could not be explored due to sample size and missing data restrictions. Hierarchical regression analyses indicated that the interaction between child shyness (at each time point) and maternal warmth and control did not predict adolescent psychopathology. Cross-lagged structural equation models analyzed the longitudinal, bidirectional relationships between child shyness and maternal warmth and control. However, youth shyness and maternal warmth/control were not correlated at any time point, youth shyness did not predict future displays of maternal warmth/control, and maternal warmth/control did not impact future levels of youth shyness. Compared to previous studies, the current study's design and methodology had many strengths. However, the findings were largely inconsistent with hypotheses and previous work. Possible explanations for these findings, study limitations, and directions for future research are summarized. / Doctor of Philosophy / The presence of psychological disorders is common during childhood and adolescence and contributes to a variety of negative outcomes. Attempts to determine what is causing these disorders to develop in youth have considered how children's temperaments and the behaviors exhibited by their parents may or may not fit well together. Past research has demonstrated that the interaction of children's fearfulness or shyness and certain parenting behaviors influences children's psychological outcomes. However, the ability to draw firm conclusions from this past research is limited by weaknesses within studies and inconsistencies between them. The current study attempted to address some of these weaknesses and inconsistencies by exploring the relationships among child shyness, maternal displays of warmth and control, and psychological symptoms. A total of 253 children had already participated in one or more assessment sessions when they were 2, 3, 4, 6, and 9-years-old, and, for the current study, 78 of those children completed an assessment as adolescents (average age = 14-years-old). Similar to previous research, at most of the ages, child shyness predicted the likelihood that the children would experience internalizing symptoms (i.e., symptoms of anxiety and mood disorders). Additionally, the way children's shyness scores changed over time also predicted which adolescents would experience internalizing symptoms. However, contrary to previous research, maternal warmth and control did not predict child psychological symptoms. Furthermore, the results did not indicate that certain parenting behaviors were better or worse for children with differing levels of shyness. This study also explored whether child shyness and maternal parenting behaviors were related to one another over time. Results indicated that shyness levels predicted future levels of shyness, and maternal warmth/control predicted future levels of warmth/control. However, again contrary to the findings of previous studies, child shyness and maternal parenting did not predict one another. Because the study findings were largely inconsistent with what was expected, possible explanations for these findings, study limitations, and directions for future research are discussed.

Yes / Aldehyde dehydrogenases (ALDHs) are overexpressed in various tumor types including prostate cancer and considered a potential target for therapeutic intervention. 4-(Diethylamino)benzaldehyde (DEAB) has been extensively reported as a pan-inhibitor of ALDH isoforms, and here, we report on the synthesis, ALDH isoform selectivity, and cellular potencies in prostate cancer cells of 40 DEAB analogues; three analogues (14, 15, and 16) showed potent inhibitory activity against ALDH1A3, and two analogues (18 and 19) showed potent inhibitory activity against ALDH3A1. Significantly, 16 analogues displayed increased cytotoxicity (IC50 = 10-200 μM) compared with DEAB (>200 μM) against three different prostate cancer cell lines. Analogues 14 and 18 were more potent than DEAB against patient-derived primary prostate tumor epithelial cells, as single agents or in combination treatment with docetaxel. In conclusion, our study supports the use of DEAB as an ALDH inhibitor but also reveals closely related analogues with increased selectivity and potency. / S12-027/PCUK_/Prostate Cancer UK/United Kingdom

Glycine receptors (GlyRs) are essential for fast inhibitory neurotransmission in the mammalian central nervous system. GlyRs form pentameric ligand-gated chloride channels and consist either of α subunits as homomers located at presynapses and extrasynaptic sites or of α and β subunits as heteromers located at the postsynapse via binding of the GlyR β subunit to the scaffold protein gephyrin. Failure in glycinergic inhibition leads to moto-neurological diseases. While mutations in genes (GLRA1, GLRB) encoding GlyR subunits are associated with hyperekplexia (startle disease), autoantibodies (aAbs) targeting GlyRs elicit the autoimmune diseases Stiff Person Syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM). In these diseases, symptoms are variable including excessive startle and stiffness in limb muscles. Hyperekplexia mutations directly interfere with ion channel functions or prevent correct receptor folding and trafficking to the membrane. In SPS, binding of GlyR aAbs leads to internalisation of receptors or directly impairs receptor function. The diseases can be treated symptomatically, but not all patients respond well to treatment options. This thesis provides further insights in hyperekplexia and SPS pathology. The involvement of the GlyR β subunit in hyperekplexia was investigated by characterisation of three novel missense mutations in GLRB: Y252S, S321F and A455P. We identified loss- or gain-of-function in combination with reduced synaptic localisation of mutated GlyRs eliciting the phenotype in patients. Furthermore, the role of unaffected GlyR β in the presence of GLRA1 mutations in mouse models for hyperekplexia carrying a missense mutation (shaky) or completely lacking GlyR α1 (oscillator) was investigated. While synaptic targeting of GlyR β was unaltered, different compensatory mechanisms were found in the mutant mice. In shaky animals, functionally impaired GlyR α1 gets transported to membranes together with GlyR β. In oscillator mice, overexpression of GlyR α2 was found to possibly compensate for the missing GlyR α1. However, compensation remains functionally insufficient in both mouse mutants. In SPS/PERM, novel targeted sites of GlyR aAbs were identified. Screening of 58 patient sera revealed autoimmune reactivity against the GlyR β subunit. Binding and specificity of aAbs to GlyR β was verified with recombinant cells, peptide microarrays, primary spinal cord neurons and tissue. Functional consequences of GlyR β aAbs differ from those of GlyR α1 aAbs. In contrast to reduced glycine potency upon binding of aAbs to GlyR α1, GlyR β aAbs decrease receptor efficacy for glycine. In addition, GlyR aAbs were shown to bind presynaptic homomeric GlyR α. We established a novel protocol to specifically culture interneurons from murine spinal cords and analysed binding of GlyR aAbs to pre- and postsynaptic sites with super-resolution microscopy. Binding to presynapses could thereby demonstrate novel insights on disease pathology mechanisms. Specification of individual targeting profiles of aAbs in SPS/PERM patients will help to further understand phenotypic differences and to develop new and more personalized treatment approaches. / Glyzin Rezeptoren (GlyR) sind essentiell für schnelle inhibitorische Neurotransmission im zentralen Nervensystem von Säugetieren. GlyR formen pentamere, Liganden-gesteuerte Chlorid Kanäle und bestehen entweder aus α Untereinheiten als Homomere lokalisiert an der Präsynapse oder extrasynaptischen Seiten oder aus α und β Untereinheiten als Heteromere lokalisiert an der Postsynapse durch Bindung von der GlyR β Untereinheit an das Gerüst-Protein Gephyrin. Versagen von glyzinerger Inhibition führt zu moto-neurologischen Krankheiten. Während Mutationen in Genen (GLRA1, GLRB) welche GlyR Untereinheiten codieren in Verbindung mit Hyperekplexie (engl. startle disease) gebracht werden, führen Autoantikörper (aAb, vom engl. autoantibody) gegen den GlyR zu den autoimmun Krankheiten Stiff Person Syndrome (SPS) und progressive Enzephalomyelitis mit Steifheit und Myoklonus (PERM, vom engl. progressive encephalomyelitis with rigidity and myoclonus). Symptome dieser Krankheiten sind variabel und beinhalten übermassiges Erschrecken und Steifheit in Muskeln der Gliedmaßen. Hyperekplexie Mutationen beeinträchtigen direkt die Ionenkanal Funktion oder verhindern die korrekte Rezeptorfaltung und den Transport zur Membran. Bindung von GlyR aAb in SPS führen zur Internalisierung der Rezeptoren oder beschädigen direkt die Rezeptorfunktion. Die Krankheiten sind symptomatisch behandelbar aber nicht alle Patienten reagieren gut auf die Behandlungsoptionen. Diese Arbeit liefert weitere Kenntnisse in der Pathologie von Hyperekplexie und SPS. Die Beteiligung der GlyR β Untereinheit in Hyperekplexie wurde untersucht indem drei neue GLRB missense-Mutationen, Y252S, S321F und A455P, charakterisiert wurden. Wir identifizierten Funktionsverlust oder -gewinn in Kombination mit reduzierter synaptischer Lokalisation von mutierten GlyR als Auslösung der Phänotypen in Patienten. Darüber hinaus untersuchten wir die Rolle von unbeeinträchtigtem GlyR β in Gegenwart von GLRA1 Mutationen in Mausmodellen für Hyperekplexie welche eine missense-Mutation (shaky) tragen oder in welchen GlyR α1 komplett fehlt (oscillator). Während die synaptische Binding von GlyR β unverändert war, fanden wir verschiedene Kompensationsmechanismen in den mutierten Mäusen. In shaky Tieren werden funktionell gestörte GlyR α1 zusammen mit GlyR β zur Membran transportiert. In oscillator Mäusen wurde eine Überexpression von GlyR α2 festgestellt welche möglicherweise das fehlende GlyR α1 kompensiert. Allerdings verbleibt die Kompensation in beiden Mäuse-Mutanten funktionell unzureichend. In SPS/PERM identifizierten wir neue Bindungsziele von GlyR aAb. Screening von 58 Patientenseren enthüllte eine autoimmune Reaktivität gegen GlyR β. Bindung und Spezifizität von aAb gegen GlyR β wurde mit rekombinanten Zellen, Peptid Mikro-arrays, primären Rückenmarksneuronen und -gewebe überprüft. Funktionelle Konsequenzen von GlyR β aAb unterschieden sich von GlyR α1 aAb. Im Kontrast zu reduzierter Wirksamkeit von Glyzin nach Bindung von aAb an GlyR α1, verringern GlyR β aAb die Effizienz von Glyzin. Zusätzlich konnten wir zeigen, dass GlyR aAb an präsynaptische, homomere GlyR α binden. Wir etablierten ein neues Protokoll, um spezifische Interneuronen-Kulturen von murinen Rückenmärkern zu kultivieren und analysierten die Bindung von GlyR aAb an prä- und postsynaptische Seiten mit hochauflösender Mikroskopie. Bindung an Präsynapsen kann dabei neue Einblicke über die Mechanismen der Krankheitspathologie aufzeigen. Spezifizierung von individuellen Bindungsprofilen von aAb in SPS/PERM Patienten wird dabei helfen die phänotypischen Unterschiede mehr zu verstehen und neue, personalisierte Behandlungsansätze zu entwickeln.

Les mécanismes d'inhibition sélective attentionnelle ont été abondamment étudiés au cours des dernières années. Les avancées en recherche ne permettent toutefois pas d'établir avec certitude s'ils sont multiples ou la manifestation d'un système unique. L'objectif de cette étude est de vérifier si l'inhibition sélective sémantique et de la localisation résultent ou non d'un mécanisme commun. Trente-et-un participants exécutent deux tâches, l'une mesurant l'effet d'amorçage négatif sémantique et l'autre évaluant l'effet d'amorçage négatif de la localisation. Des effets d'inhibition sont obtenus aux deux procédures. Les résultats observés indiquent cependant l'absence de relation statistique entre les mesures d'inhibition sémantique et spatiale. Les résultats sont discutés en rapport avec la littérature pertinente dans le domaine.

Previous studies on inhibition of the Escherichia coli glycerol kinase enzyme have suggested that subunit-subunit or domain bridging interactions form part of the network in communicating ligand binding to inhibition. In this study, five amino acids were identified to be in close proximity to an Arg369 residue which is a domain bridging residue. Three of the amino acid residues (Q37, Y39 and Q104) are in domain I of the enzyme subunit, while the other two (M308 and Q314) are in domain II of the enzyme subunit. To evaluate the importance of each domain bridging residue in IIAGlc inhibition, alanine substitutions were made of the residues, and the kinetic properties characterized with respect to IIAGlc inhibition. Kinetic parameters obtained for each variant glycerol kinase enzyme was compared to values obtained for the Wild Type enzyme to assess the importance of the amino acid residue in IIAGlc inhibition. The effects of the substitutions on FBP inhibition as well as catalysis of the enzyme were also analyzed by obtaining kinetic parameters for each of the variant enzymes. The results from this study indicate that the domain I bridging interactions with Arg369 are important in IIAGlc regulation of the E. coli glycerol kinase enzyme. The domain II bridging interactions appear to be unimportant in regulating IIAGlc inhibition. Two of the domain I bridging residues studied were also found to be important in FBP inhibition. These results indicate that some the domain bridging residues seen to be involved in IIAGlc regulation also appear to be involved in FBP regulation. In catalysis, with the exception of Q314, the rest of the domain I and II bridging residues appear to be important for substrate binding and/or catalysis.

The aim of this experimental study is to investigate the inhibitive properties of polyglycol and polyglycol+KCl aqueous solutions on hydrate formation, which causes serious fluid flow problems, especially during deepwater drilling operations. As the petroleum industry continues to search oil in deeper and deeper seas, the possibility of facing hydrate problems during drilling operations increases because of the suitable conditions for hydrate formation. The main goal of this study is to investigate the hydrate inhibition capacity (thermodynamic and/or kinetic inhibition) of polyglycol and KCl which are mainly used in drilling fluids for shale inhibition and wellbore stability. A high pressure hydrate forming reactor is used to form and dissociate methane hydrate from aqueous solutions of polyglycol and polyglycol+KCl. In total 10 experiments were carried out, 5 of them with 0%, 1%, 3%, 5% and 7 % by volume of polyglycol solutions (Group-A experiments). The remaining 5 experiments (Group-B) had 8% by weight of KCl in solution in addition to the same polyglycol concentrations of Group-A experiments. Among the two chemicals tested for their hydrate inhibiting potentials, polyglycol did not exhibit any thermodynamic inhibition capacity while KCl was observed to have the ability of hydrate inhibition thermodynamically. On the other hand, increase in polyglycol concentration at constant KCl concentration (Group-B) increases the hydrate formation depression capacity of KCl. Polyglycol inhibits methane hydrate formation kinetically. The higher the polyglycol concentration in aqueous solution, the lower is the initial rate of methane hydrate formation (corresponding to first 15 minutes of hydrate formation). On the other hand, there exists a slower change of methane hydrate formation rate as polyglycol concentration increases.